Slow decrease in temperature produces readthrough transcripts in mammalian hibernation.

Accumulating evidence suggests that various cellular stresses interfere with the end processing of mRNA synthesis and lead to the production of abnormally long transcripts, known as readthrough transcripts (RTTs), which extend beyond the termination sites. Small mammalian hibernators repeatedly enter a state referred to as deep torpor (DT), where the metabolic rate, respiration rate, and core body temperature become extremely low, which produces various types of cellular stresses and therefore induces RTTs. However, the types of stresses and processes around the DT that cause RTTs are unclear. In the present study, we showed that RTTs are produced from different gene loci in the livers of Syrian hamsters under DT and summer-like conditions. Moreover, in vitro analysis using hamster primary hepatocytes revealed that DT-specific RTTs are induced by a slow decline in temperature, as seen in body temperature in the entrance phase of DT, but not by rapid cold treatment or hypoxia. In addition, it was observed that RTTs were not elongated under a significantly cold temperature (4 °C). These results indicate that DT-specific RTTs are produced during the entrance phase of torpor by a slow decrease in body temperature.

Spontaneous recurrence of a summer-like diel rhythm in the body temperature of the Syrian hamster after hibernation.

Mammalian hibernation is a survival strategy characterized by metabolic suppression and drastically lowering body temperature (Tb), used during harsh seasons with food shortages and cold. The Syrian hamster commences hibernation in response to a short photoperiod and cold but spontaneously concludes hibernation after several months without environmental cues. Little is known about the changes in diel rhythms during hibernation. Using long-term and high-resolution Tb data, we analysed the diel Tb rhythm time-course changes in Syrian hamsters raised under summer-like conditions (long photoperiod (LP) and warm; LP-warm) and transferred to winter-like conditions (short photoperiod (SP) and cold; SP-cold). The diel Tb rhythm was undetectable during the hibernation period (HIBP), reappearing after the HIBP. The phase of this returning rhythm reverted to the LP entrainment phase characteristics despite the ambient SP and then re-entrained to the ambient SP as if the hamsters were transferred from the LP-warm to SP-cold conditions. The diel Tb rhythm reverted from the SP- to LP-type in a hibernation-dependent manner. Under constant dark and cold conditions, the circadian Tb rhythm recovered without photic stimuli following the HIBP. These findings suggest that hibernation involves a program that anticipates the ambient photoperiod when animals emerge from hibernation.

Step-by-step protocols for non-viral derivation of transgene-free induced pluripotent stem cells from somatic fibroblasts of multiple mammalian species.

Potentials of immortal proliferation and unlimited differentiation into all the three germ layers and germ cells in induced pluripotent stem cells (iPSCs) render them important bioresources for in vitro reconstitution and modeling of intravital tissues and organs in various animal models, thus contributing to the elucidation of pathomechanisms, drug discovery and stem cell-based regenerative medicine. We previously reported promising approaches for deriving transgene-free iPSCs from somatic fibroblasts of multiple mammalian species by episomal vector or RNA transfection, although the respective step-by-step protocols and the combinatorial usage of these methods, which achieved high induction efficiency, have not been described in the literature so far. Here, we provide a detailed step-by-step description of these methods with critical tips and slight modifications (improvements) to previously reported methods. We also report a novel method for the establishment of iPSCs from the Syrian hamster (also known as golden hamster; Mesocricetus auratus), a unique animal model of hibernation. We anticipate this methodology will contribute to stem cell biology and regenerative medicine research.

Apoptosis is involved in maintaining the character of the midbrain and the diencephalon roof plate after neural tube closure.

Apoptosis, a major form of programmed cell death, is massively observed in neural plate border and subsequently in the roof plate (RP). While deficiency of apoptosis often results in brain malformations including exencephaly and hydrocephalus, the impact of apoptosis on RP formation and maintenance remains unclear. Here we described that mouse embryos deficient in Apaf1, a gene crucial for the intrinsic apoptotic pathway, in C57BL/6 genetic background exhibited narrow and discontinuous expression of RP marker genes in the midline of the midbrain and the diencephalon. Instead, cells positive for the neuroectodermal gene SOX1 ectopically accumulated in the midline. A lineage-tracing experiment suggests that these ectopic SOX1-positive cells began to accumulate in the midline of apoptosis-deficient embryos after E9.5. These embryos further displayed malformation of the subcommissural organ, which has been discussed in the etiology of hydrocephalus. Thus, the apoptosis machinery prevents ectopic emergence of SOX1-positive cells in the midbrain and the diencephalon RP, and helps in maintaining the character of the RP in the diencephalon and midbrain, thereby ensuring proper brain development.

Caspase-3 regulates ureteric branching in mice via cell migration.

Inhibition of caspase-3 (Casp3) reduces ureteric branching in organ culture but the mechanism remains unclear. Since Casp3 has non-apoptotic functions, we examined whether Casp3 regulates ureteric branching by promoting cell migration, using a ureteric bud (UB) cell line and Casp3-deficient (Casp3-/-) mice. Also, we examined whether Casp3 plays a role in the reduced ureteric branching of metanephroi from nutrient restricted mothers, in which Casp3 activity is suppressed. A Casp3 inhibitor Ac-DNLD-CHO reduced FGF2-induced cord formation of UB cells in 3D culture. UB cell migration assessed by Boyden chamber and wound healing assays was inhibited by Ac-DNLD-CHO. Glomerular number was reduced by ≈ 30%, and ureteric tip number was lower in Casp3-/- mice compared with controls. Maternal nutrient restriction decreased ureteric tip number in controls but not in Casp3-/-. In conclusion, Casp3 regulates ureteric branching by promoting UB cell migration. Inhibited ureteric branching by maternal nutrient restriction may be mediated by Casp3.

Rewiring of embryonic glucose metabolism via suppression of PFK-1 and aldolase during mouse chorioallantoic branching.

Adapting the energy metabolism state to changing bioenergetic demands is essential for mammalian development accompanying massive cell proliferation and cell differentiation. However, it remains unclear how developing embryos meet the changing bioenergetic demands during the chorioallantoic branching (CB) stage, when the maternal-fetal exchange of gases and nutrients is promoted. In this study, using metabolome analysis with mass-labeled glucose, we found that developing embryos redirected glucose carbon flow into the pentose phosphate pathway via suppression of the key glycolytic enzymes PFK-1 and aldolase during CB. Concomitantly, embryos exhibited an increase in lactate pool size and in the fractional contribution of glycolysis to lactate biosynthesis. Imaging mass spectrometry visualized lactate-rich tissues, such as the dorsal or posterior neural tube, somites and head mesenchyme. Furthermore, we found that the heterochronic gene Lin28a could act as a regulator of the metabolic changes observed during CB. Perturbation of glucose metabolism rewiring by suppressing Lin28a downregulation resulted in perinatal lethality. Thus, our work demonstrates that developing embryos rewire glucose metabolism following CB for normal development.

Loss of the small GTPase Arl8b results in abnormal development of the roof plate in mouse embryos.

Lysosomes are acidic organelles responsible for degrading both exogenous and endogenous materials. The small GTPase Arl8 localizes primarily to lysosomes and is involved in lysosomal function. In the present study, using Arl8b gene-trapped mutant (Arl8b-/- ) mice, we show that Arl8b is required for the development of dorsal structures of the neural tube, including the thalamus and hippocampus. In embryonic day (E) 10.5 Arl8b-/- embryos, Sox1 (a neuroepithelium marker) was ectopically expressed in the roof plate, whereas the expression of Gdf7 and Msx1 (roof plate markers) was reduced in the dorsal midline of the midbrain. Ectopic expression of Sox1 in Arl8b-/- embryos was detected also at E9.0 in the neural fold, which gives rise to the roof plate. In addition, the levels of Bmp receptor IA and phosphorylated Smad 1/5/8 (downstream of BMP signaling) were increased in the neural fold of E9.0 Arl8b-/- embryos. These results suggest that Arl8b is involved in the development of the neural fold and the subsequently formed roof plate, possibly via control of BMP signaling.

Temporal regulation of Lin28a during mammalian neurulation contributes to neonatal body size control.

BACKGROUND: The timing of developmental events is tightly regulated along a time axis for normal development. Although the RNA-binding protein Lin28a plays a crucial role in the regulation of developmental timing in Caenorhabditis elegans, how the timing of Lin28a expression affects the rate and/or duration of developmental events during mammalian development remains to be addressed. RESULTS: In this study, we discovered that the timing and the duration of Lin28a expression affect embryonic growth. During the neurulation stage of mouse development, endogenous Lin28a levels start to drop. When Lin28a expression was maintained transiently using the inducible tetracycline-regulated gene expression (Tet-ON) system [doxycycline (Dox)-inducible Lin28a transgenic (iLin28a Tg) mice] with Dox administration at E8.5 and E9.5, it resulted in neonatal lethality, increased body weight (organomegaly), and an increased number of caudal vertebrae at birth. On the other hand, Lin28a induction only at E8.5 caused neonatal lethality and organomegaly, but did not affect the caudal vertebra number. Of note, although Dox treatment before or after neurulation still caused neonatal lethality, it neither caused organomegaly nor the increased caudal vertebra number in iLin28a Tg neonates. CONCLUSIONS: Temporal regulation of Lin28a expression during neurulation affects developmental events such as cessation of axial elongation and embryonic growth in mice.

A Fluorescent Probe for Rapid, High-Contrast Visualization of Folate-Receptor-Expressing Tumors In†Vivo.

Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR-α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near-infrared (NIR) region (650-900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR-α show high non-specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR-1, utilizing a Si-rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor-to-background ratio (TBR) of up to 83 in FR-expressing tumor-bearing mice within 30 min. Thus, FolateSiR-1 has the potential to contribute to the research in the field of biology and the clinical medicine.

Arl8b is required for lysosomal degradation of maternal proteins in the visceral yolk sac endoderm of mouse embryos.

The small GTPase Arl8b localizes primarily to lysosomes and is involved in lysosomal motility and fusion. Here, we show that Arl8b is required for lysosomal degradation of maternal proteins in the visceral yolk sac endoderm (VYSE), an apical cell layer of the visceral yolk sac, of mouse embryos. The VYSE actively takes up maternal materials from uterine fluid and degrades them in lysosomes to provide breakdown products to the embryo. Arl8b gene-trap mice (Arl8b-/- ) displayed decreased early embryo body size. The Arl8b-/-  VYSE exhibited defective endocytic trafficking to the lysosome and accumulation of maternal proteins such as albumin and immunoglobulin G in late endocytic organelles. Furthermore, Transthyretin-Cre;Arl8bflox/flox mice in which Arl8b was ablated specifically in the VYSE also showed decreased embryo body size, defects in trafficking to the lysosome and reduction of the free amino acid level in the embryos. Taken together, these results suggest that Arl8b mediates lysosomal degradation of maternal proteins in the VYSE, thereby contributing to mouse embryonic development.

Mammalian embryos show metabolic plasticity toward the surrounding environment during neural tube closure.

Developing embryos rewire energy metabolism for developmental processes. However, little is known about how metabolic rewiring is coupled with development in a spatiotemporal manner. Here, we show that mammalian embryos display plasticity of glucose metabolism in response to the extracellular environment at the neural tube closure (NTC) stage, when the intrauterine environment changes upon placentation. To study how embryos modulate their metabolic state upon environmental change, we analyzed the steady-state level of ATP upon exposure to extrauterine environments using both an enzymatic assay and a genetically encoded ATP sensor. Upon environmental changes, NTC-stage embryos exhibited increased ATP content, whereas embryos before and after NTC did not. The increased ATP in the NTC-stage embryos seemed to depend on glycolysis. Intriguingly, an increase in mitochondrial membrane potential (ΔΨm) was also observed in the neural ectoderm (NE) and the neural plate border of the non-neural ectoderm (NNE) region. This implies that glycolysis can be coupled with the TCA cycle in the NE and the neural plate border depending on environmental context. Disrupting ΔΨm inhibited folding of the cranial neural plate. Thus, we propose that embryos tune metabolic plasticity to enable coupling of glucose metabolism with the extracellular environment at the NTC stage.

Caspases and matrix metalloproteases facilitate collective behavior of non-neural ectoderm after hindbrain neuropore closure.

BACKGROUND: Mammalian brain is formed through neural tube closure (NTC), wherein both ridges of opposing neural folds are fused in the midline and remodeled in the roof plate of the neural tube and overlying non-neural ectodermal layer. Apoptosis is widely observed from the beginning of NTC at the neural ridges and is crucial for the proper progression of NTC, but its role after the closure remains less clear. RESULTS: Here, we conducted live-imaging analysis of the mid-hindbrain neuropore (MHNP) closure and revealed unexpected collective behavior of cells surrounding the MHNP. The cells first gathered to the closing point and subsequently relocated as if they were released from the point. Inhibition of caspases or matrix metalloproteases with chemical inhibitors impaired the cell relocation. CONCLUSIONS: These lines of evidence suggest that apoptosis-mediated degradation of extracellular matrix might facilitate the final process of neuropore closure.

Detection of LacZ-Positive Cells in Living Tissue with Single-Cell Resolution.

The LacZ gene, which encodes Escherichia coli ß-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic ß-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.

How to form and close the brain: insight into the mechanism of cranial neural tube closure in mammals.

The development of the embryonic brain critically depends on successfully completing cranial neural tube closure (NTC). Failure to properly close the neural tube results in significant and potentially lethal neural tube defects (NTDs). We believe these malformations are caused by disruptions in normal developmental programs such as those involved in neural plate morphogenesis and patterning, tissue fusion, and coordinated cell behaviors. Cranial NTDs include anencephaly and craniorachischisis, both lethal human birth defects. Newly emerging methods for molecular and cellular analysis offer a deeper understanding of not only the developmental NTC program itself but also mechanical and kinetic aspects of closure that may contribute to cranial NTDs. Clarifying the underlying mechanisms involved in NTC and how they relate to the onset of specific NTDs in various experimental models may help us develop novel intervention strategies to prevent NTDs.

Cold resistance of mammalian hibernators ∼ a matter of ferroptosis?

Most mammals adapt thermal physiology around 37°C and large deviations from their range, as observed in severe hypothermia and hyperthermia, resulting in organ dysfunction and individual death. A prominent exception is mammalian hibernation. Mammalian hibernators resist the long-term duration of severe low body temperature that is lethal to non-hibernators, including humans and mice. This cold resistance is supported, at least in part, by intrinsic cellular properties, since primary or immortalized cells from several hibernator species can survive longer than those from non-hibernators when cultured at cold temperatures. Recent studies have suggested that cold-induced cell death fulfills the hallmarks of ferroptosis, a type of necrotic cell death that accompanies extensive lipid peroxidation by iron-ion-mediated reactions. In this review, we summarize the current knowledge of cold resistance of mammalian hibernators at the cellular and molecular levels to organ and systemic levels and discuss key pathways that confer cold resistance in mammals.

Nail growth arrest under low body temperature during hibernation.

Growth and differentiation are reduced or stopped during hibernation, an energy conserving strategy in harsh seasons by lowered metabolism and body temperature. However, few studies evaluated this in a same individual using a non-invasive method. In this study, we applied a non-invasive tracking method of the nail growth throughout the hibernation period in the same hibernating animals, the Syrian hamster (Mesocricetus auratus). We found that nail growth was markedly suppressed during the hibernation period but rapidly recovered by the exit from the hibernation period. Our data suggest that nail growth was arrested during deep torpor, a hypometabolic and hypothermic state, but recovered during periodic arousal, a euthermic phase. Consistent with this, nail stem cells located in the nail matrix did not exit the cell cycle in the deep torpor. Thus, hibernation stops nail growth in a body temperature-dependent manner.

Text mining online disinformation about antihypertensive agents ACEI/ARB and COVID-19 on Sina Weibo.

Background: The global COVID-19 pandemic outbreak has caused a significant social and economic burden, with over 4.7 million confirmed cases and thousands of casualties. Moreover, pandemic-related misinformation and disinformation on social media platforms have led to intense psychosocial issues. We investigated online disinformation about angiotensin-converting enzyme inhibitors (ACEI)/angiotensin receptor blocker (ARB) drugs and their relationship to COVID-19 on Sina Weibo. Methods: We searched for posts related to the pandemic from its beginning in December 2019 to 19 January 2021. We used text mining to identify content related to "antihypertensive agents ACEI/ARB can increase COVID-19". Results: We found 82 posts spreading disinformation and 44 posts dispelling disinformation. The former had 535 clicks and concerns and 31 comments, and was forwarded 98 times. Of the 82 posts spreading disinformation, 15.9% (n = 13) contained pseudo-scientific information, 24.4% (n = 20) contained authoritative releases, and 75.6% (n = 62) contained normal personal releases. Most disinformation posts (n = 61 (74.3%)) were published from 16 February 2020 to 16 March 2020, and 12.2% (n = 10) were published from 1 February 2021 to 16 March 2021. Among the 44 dispelling disinformation posts, approximately 57.1% of the comments were in support, and 42.9% were opposed or invalid. Nearly half of the users were confused or superstitious about the disinformation. Conclusions: The disinformation about ACEI/ARB increasing the opportunity for COVID-19 infection during the pandemic was based on clinical mechanisms and scientific evidence intended for hypertensive patients taking long-term medication. It was packaged in a pseudo-scientific shell, leading to confusion and panic among patients. This disinformation harmed COVID-19 prevention efforts, damaged mental health, and possibly led to harmful behaviours. In future crises, the spread of rumours should be stopped quickly and effectively.

Cold-induced suspension and resetting of Ca2+ and transcriptional rhythms in the suprachiasmatic nucleus neurons.

Does the circadian clock keep running under such hypothermic states as daily torpor and hibernation? This fundamental question has been a research subject for decades but has remained unsettled. We addressed this subject by monitoring the circadian rhythm of clock gene transcription and intracellular Ca2+ in the neurons of the suprachiasmatic nucleus (SCN), master circadian clock, in vitro under a cold environment. We discovered that the transcriptional and Ca2+ rhythms are maintained at 22°C-28°C, but suspended at 15°C, accompanied by a large Ca2+ increase. Rewarming instantly resets the Ca2+ rhythms, while transcriptional rhythms reach a stable phase after the transient state and recover their phase relationship with the Ca2+ rhythm. We conclude that SCN neurons remain functional under moderate hypothermia but stop ticking in deep hypothermia and that the rhythms reset after rewarming. These data also indicate that stable Ca2+ oscillation precedes clock gene transcriptional rhythms in SCN neurons.

Generation of transgenic mice expressing a FRET biosensor, SMART, that responds to necroptosis.

Necroptosis is a regulated form of cell death involved in various pathological conditions, including ischemic reperfusion injuries, virus infections, and drug-induced tissue injuries. However, it is not fully understood when and where necroptosis occurs in vivo. We previously generated a Forster resonance energy transfer (FRET) biosensor, termed SMART (the sensor for MLKL activation by RIPK3 based on FRET), which monitors conformational changes of MLKL along with progression of necroptosis in human and murine cell lines in vitro. Here, we generate transgenic (Tg) mice that express the SMART biosensor in various tissues. The FRET ratio is increased in necroptosis, but not apoptosis or pyroptosis, in primary cells. Moreover, the FRET signals are elevated in renal tubular cells of cisplatin-treated SMART Tg mice compared to untreated SMART Tg mice. Together, SMART Tg mice may provide a valuable tool for monitoring necroptosis in different types of cells in vitro and in vivo.

Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system.

In utero electroporation is widely used to study neuronal development and function by introducing plasmid DNA into neural progenitors during embryogenesis. This is an effective and convenient method of introducing plasmid DNA into neural precursors and is suitable for manipulating gene expression in cells of the CNS. However, the applicability of this technique is comparatively limited to neuronal research, as the plasmid DNA introduced into neural progenitors during embryogenesis is diluted by cell proliferation and is not stably maintained in glial cells generated around and after birth. To overcome this limitation, we applied the Tol2 transposon system, which integrates a transgene into the genome of the host cell, to in utero electroporation. With this system, we confirmed that the transgene was effectively maintained in the progeny of embryonic neural precursors, astrocytes and oligodendrocytes. Using the glial promoters GFAP and S100beta, targeted and stable expressions of transgenes in glia were obtained, which enabled the expression of different transgenes simultaneously in neurons and glia. Glia-targeted expression of the transgene that causes neuronal migration defect was achieved without the defect. Thus, use of the Tol2 transposon system in combination with in utero electroporation is a powerful method for studying glia-neuron interactions in vivo.